Phylogenetic relationship of Plasmodium falciparum populations in the Philippines.

Malaria is one of the major infectious diseases in the Philippines. It is being targeted for control through sustained early diagnosis, treatment and mosquito control. It is in this light that understanding the genetic background of the parasite population is important not only for basic biology of the organism but also for epidemiology and control of the disease. In the present study, molecular phylogenetic relationships of the 3 Plasmodium falciparum populations in the Philippines with the other populations in the world were inferred based on polymorphisms of 9 highly polymorphic microsatellite DNA loci in the parasite genome. A total of 92 P. falciparum isolates collected from 3 provinces (Kalinga, Palawan and Davao del Norte) in the Philippines, and 8 from other populations (3 African, 2 South American, 2 Papua New Guinean, and 1 Thai) that were previously reported, were used for the analysis. The phylogenetic tree showed that the 3 Philippine populations were genetically divergent from each other as compared to the other populations. The branching pattern of the tree suggests that the 3 Philippine populations were relatively close to the Thai population, rather than the Papua New Guinean populations, indicating that the ancestor of the 3 Philippines populations were introduced from Indochina peninsula, and not from countries located south of the Philippines such as Papua New Guinea or Indonesia.

In vitro susceptibility of Plasmodium falciparum isolates to chloroquine and mefloquine in southeastern Mindanao Island, the Philippines.

Although the presence of multi-drug-resistant falciparum malaria has been reported in the Philippines, the distribution of drug-resistant malaria parasites has not yet been determined in Mindanao Island. In vitro susceptibility of P. falciparum to both chloroquine and mefloquine was assessed to forecast the spread of drug-resistant parasites in various foci in southeastern Mindanao Island. Of the 33 isolates of P. falciparum successfully tested, 10 (30%) were susceptible, 12 (36%) showed decreased susceptibility (80 nM < or = IC50 < 114 nM), and 11 (33%) were resistant (IC50 > or = 114 nM) to chloroquine. Ten (91%) of the resistant isolates and 9 (75%) of those with decreased susceptibility were from northern and northwestern Davao del Norte Province. Chloroquine-susceptible isolates were found among patients in the eastern parts of Davao del Norte and Davao Oriental provinces. Seven isolates from several foci in the study area were all mefloquine- susceptible (IC50 < 10 nM). This is the first report indicating the potential emergence of chloroquine-resistant P. falciparum on Mindanao Island, which is presently regarded as a drug-susceptible area.

PCR-amplification, sequencing, and comparison of the var/PfEMP-1 gene from the blood of patients with falciparum malaria in the Philippines.

The adhesion of Plasmodium falciparum-infected erythrocytes to vascular endothelium and to uninfected red blood cells (RBCs) plays a key role in the pathology of severe malaria. Adhesion is known to be mediated in part by the antigenically-variant erythrocyte membrane protein-1 (PfEMP-1), which is encoded by the var-gene family of P. falciparum. It has recently been reported that in vitro a single parasite simultaneously transcribes multiple var-genes but that, through a developmentally regulated process, the parasite selects only one PfEMP-1 that will to reach the surface of the host RBC. Were this to be true in vivo, one would expect a correlation between the type of var/PfEMP-1 that is expressed on the parasite-infected RBC and the severity of clinical disease. In order to test this assumption, we determined the sequence of the var-gene that was expressed by the parasites in patients' blood samples. Seven blood samples were collected from patients with or without severe clinical symptoms (cerebral malaria): two samples were from patients diagnosed as having imported falciparum malaria at the International Medical Center of Japan (IMCJ); the five others were from patients of the Davao Regional Hospital in Davao, the Philippines. The parasites (ring stage) in the blood samples were cultured for 24 hours; the matured trophozoites, in which the var-gene selection had taken place, served as material for mRNA isolation. The cDNA corresponding to the Duffy-binding-like (DBL)-1 domain of the var-gene was amplified by RT-PCR, using a region-specific primer set. The amplified cDNAs were cloned into the plasmid vector; the resultant clones (32) were sequenced on both strands. The results indicated that there was considerable diversity in the sequence of the DBL-1 domain among the clones, even among those from a single patient. In conclusion, it was difficult to demonstrate the correlation between the type of var-gene transcripts found in the RBCs of malaria patients and the severity of their symptoms.

A study of chloroquine resistance of plasmodium falciparum using the in-vitro sensitivity test and polymerase chain reaction (PCR)

The widespread and increasing resistance of Plasmodium falciparum to antimalarial drugs is one of several factors that contributed to the persistence and even worsening of the malaria problem. Resistance to Chloroquine is of utmost concern, considering that it had been the most reliable antimalarial until the emergence and spread of Chloroquineresistant P. falciparum. Until recently, Chloroquine and Sulfadoxine-Pyrimethamine were the first and second line antimalarials in use in the Philippines. However, this has been changed to a combination of Chloroquine and Sulfadoxine-Pyrimethamine, because of the high percentage of treatment failures in therapeutic efficacy studies done in northern and southern Philippines. The objective of this study is to apply PCR in determining the occurence of Chloroquine resistance in southeastern Mindanao using in-vitro test and polymerase chain reaction (PCR)In the first study, the in-vitro susceptibility of P. falciparum to Chloroquine was tested in 33 isolates using the World Health Organization (WHO) Semi-Microtest Method. These isolates were collected from patients who consulted or were admitted at the regional hospital of Davao del Norte. The results showed that 10 (30.3 percent) were susceptible with IC50 80 nM, 12 (36.4 percent) isolates had decreased sensitivity with 80 nM /- IC50 114 nM, and 11 (33.3 percent) were resistant with IC 114 nM. Ten of these 11 (91 percent) were from Davao del Norte. A closer look at the municipalities of this province showed that the geometric mean (SD) of IC50 of Asuncion was 133 (41) nM and was significantly higher (p=0.025) than nearby Kapalong at 82 (25) nMThe PCR, Apo1 restriction revealed that 30 (90.9 percent) of the isolates manifested the PfCRT (K76T) mutation. These findings are indicative of the presence of Chloroquine resistance among the isolates. Comparison with the results of the invitro test (33.3 percent resistance) showed that the frequency of the PfCRT gene (90.9 percent) was very high. This finding suggests that the mere presence of the PfCRT gene does not mean the expression of Chloroquine resistance. It is possible that other genes such as the Pfmdr and cg2 are also involved in the expression of Chloroquine resistance. The study also shows that PCR and Apo1 restriction may be limited in generating results that can be used to correlate with those of the in-vitro or even in-vivo tests